Pet127p, a membrane-associated protein involved in stability and processing of Saccharomyces cerevisiae mitochondrial RNAs.

Nuclear mutations that inactivate the Saccharomyces cerevisiae gene PET127 dramatically increased the levels of mutant COX3 and COX2 mitochondrial mRNAs that were destabilized by mutations in their 5' untranslated leaders. The stabilizing effect of pet127 delta mutations occurred both in the presence and in the absence of translation. In addition, pet127 delta mutations had pleiotropic effects on the stability and 5' end processing of some wild-type mRNAs and the 15S rRNA but produced only a leaky nonrespiratory phenotype at 37 degrees C. Overexpression of PET127 completely blocked respiratory growth and caused cells to lose wild-type mitochondrial DNA, suggesting that too much Pet127p prevents mitochondrial gene expression. Epitope-tagged Pet127p was specifically located in mitochondria and associated with membranes. These findings suggest that Pet127p plays a role in RNA surveillance and/or RNA processing and that these functions may be membrane bound in yeast mitochondria.     

Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance

In yeast the UPF1, UPF2 and UPF3 genes encode three interacting factors involved in translation termination and nonsense-mediated mRNA decay (NMD). UPF1 plays a central role in both processes. In addition, UPF1 was originally isolated as a multicopy suppressor of mitochondrial splicing deficiency, and its deletion leads to an impairment in respiratory growth. Here, we provide evidence that inactivation of UPF2 or UPF3, like that of UPF1, leads to an impairment in respiratory competence, suggesting that their products, Upf1p, Upf2p and Upf3p, are equivalently involved in mitochondrial biogenesis. In addition, however, we show that only Upf1p acts as a multicopy suppressor of mitochondrial splicing deficiency, and its activity does not require either Upf2p or Upf3p. Mutations in the conserved cysteine- and histidine-rich regions and ATPase and helicase motifs of Upf1p separate the ability of Upf1p to complement the respiratory impairment of a Deltaupf1 strain from its ability to act as a multicopy suppressor of mitochondrial splicing deficiency, indicating that distinct pathways express these phenotypes. In addition, we show that, when overexpressed, Upf1p is not detected within mitochondria, suggesting that its role as multicopy suppressor of mitochondrial splicing deficiency is indirect. Furthermore, we provide evidence that cells overexpressing certain upf1 alleles accumulate a phosphorylated isoform of Upf1p. Altogether, these results indicate that overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance, which relies on Upf1p-Upf2p-Upf3p functional interplay.     

Slx9p facilitates efficient ITS1 processing of pre-rRNA in Saccharomyces cerevisiae

Slx9p (Ygr081cp) is a nonessential yeast protein previously linked genetically with the DNA helicase Sgs1p. Here we report that Slx9p is involved in ribosome biogenesis in the yeast Saccharomyces cerevisiae. Deletion of SLX9 results in a mild growth defect and a reduction in the level of 18S rRNA. Co-immunoprecipitation experiments showed that Slx9p is associated with 35S, 23S, and 20S pre-rRNA, as well as U3 snoRNA and, thus, is a bona fide component of pre-ribosomes. The most striking effects on pre-rRNA processing resulting from deletion of SLX9 is the accumulation of the mutually exclusive 21S and 27SA2 pre-rRNA. Furthermore, deletion of SLX9 is synthetically lethal with mutations in Rrp5p that block cleavage at either site A2 or A3. We conclude that Slx9p has a unique role in the processing events responsible for separating the 66S and 43S pre-ribosomal particles. Interestingly, homologs of Slx9p were found only in other yeast species, indicating that the protein has been considerably less well conserved during evolution than the majority of trans-acting processing factors.     

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.     

Absence of the mitochondrial AAA protease Yme1p restores F0-ATPase subunit accumulation in an oxa1 deletion mutant of Saccharomyces cerevisiae

The nuclear gene OXA1 encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome c oxidase (Cox) and ATPase. In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery. We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes in Deltaoxa1, Deltaoxa1Deltaafg3, and Deltaoxa1Deltayme1 mutants. We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p. Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits. However, the F(0) subunits Atp4p, Atp6p, and Atp17p are stabilized in the Deltaoxa1Deltayme1 double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p. These results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable in Saccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3 and YME1 is lethal and that the essential function does not reside in their protease activity.     

Translation initiation in Saccharomyces cerevisiae mitochondria: functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase and novel protein Rmd9p

Rsm28p is a dispensable component of the mitochondrial ribosomal small subunit in Saccharomyces cerevisiae that is not related to known proteins found in bacteria. It was identified as a dominant suppressor of certain mitochondrial mutations that reduced translation of the COX2 mRNA. To explore further the function of Rsm28p, we isolated mutations in other genes that caused a synthetic respiratory defective phenotype together with rsm28Delta. These mutations identified three nuclear genes: IFM1, which encodes the mitochondrial translation initiation factor 2 (IF2); FMT1, which encodes the methionyl-tRNA-formyltransferase; and RMD9, a gene of unknown function. The observed genetic interactions strongly suggest that the ribosomal protein Rsm28p and Ifm1p (IF2) have similar and partially overlapping functions in yeast mitochondrial translation initiation. Rmd9p, bearing a TAP-tag, was localized to mitochondria and exhibited roughly equal distribution in soluble and membrane-bound fractions. A small fraction of the Rmd9-TAP sedimented together with presumed monosomes, but not with either individual ribosomal subunit. Thus, Rmd9 is not a ribosomal protein, but may be a novel factor associated with initiating monosomes. The poorly respiring rsm28Delta, rmd9-V363I double mutant did not have a strong translation-defective phenotype, suggesting that Rmd9p may function upstream of translation initiation, perhaps at the level of localization of mitochondrially coded mRNAs.     

Requirements of Cyc2p and the porin, Por1p, for ionic stability and mitochondrial integrity in Saccharomyces cerevisiae

It was previously demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein; that the cyc2-Delta2 deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c; that the deletion causes a defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources; and that this defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a nonionic compound. We report that por1-Delta strains lacking the mitochondrial porin, Por1p, but not por2-Delta strains lacking the related porin, share some phenotypes similar to the cyc2-Delta2 strain, including hypersensitivity to KCl in glycerol medium. Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Delta2 strain, while swelling could be detected in mitochondria from the other strains only after the addition of KCl. Thus, highly unspecific membrane permeation may be triggered by ATP in the cyc2-Delta2 strain. We suggest that Por1p and Cyc2p, in addition to their own unique functions, serve to maintain the osmotic stability of mitochondria, but by different mechanisms.     

Effects of camptothecin or TOP1 overexpression on genetic stability in Saccharomyces cerevisiae

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation.     

Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae

Mutations in SURF1, the human homologue of yeast SHY1, are responsible for Leigh's syndrome, a neuropathy associated with cytochrome oxidase (COX) deficiency. Previous studies of the yeast model of this disease showed that mutant forms of Mss51p, a translational activator of COX1 mRNA, partially rescue the COX deficiency of shy1 mutants by restoring normal synthesis of the mitochondrially encoded Cox1p subunit of COX. Here we present evidence showing that Cox1p synthesis is reduced in most COX mutants but is restored to that of wild type by the same mss51 mutation that suppresses shy1 mutants. An important exception is a null mutation in COX14, which by itself or in combination with other COX mutations does not affect Cox1p synthesis. Cox14p and Mss51p are shown to interact with newly synthesized Cox1p and with each other. We propose that the interaction of Mss51p and Cox14p with Cox1p to form a transient Cox14p-Cox1p-Mss51p complex functions to downregulate Cox1p synthesis. The release of Mss51p from the complex occurs at a downstream step in the assembly pathway, probably catalyzed by Shy1p.     

木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes.     

HFA1 encoding an organelle-specific acetyl-CoA carboxylase controls mitochondrial fatty acid synthesis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae gene, HFA1, encodes a >250-kDa protein, which is required for mitochondrial function. Hfa1p exhibits 72% overall sequence similarity (54% identity) to ACC1-encoded yeast cytoplasmic acetyl-CoA carboxylase. Nevertheless, HFA1 and ACC1 functions are not overlapping because mutants of the two genes have different phenotypes and do not complement each other. Whereas ACC1 is involved in cytoplasmic fatty acid synthesis, the phenotype of hfa1Delta disruptants resembles that of mitochondrial fatty-acid synthase mutants. They fail to grow on lactate or glycerol, and the mitochondrial cofactor, lipoic acid, is reduced to <10% of its normal cellular concentration. Other than Acc1p, the N-terminal sequence of Hfa1p comprises a canonical mitochondrial targeting signal together with a matrix protease cleavage site. Accordingly, the HFA1-encoded protein was specifically assigned by Western blotting of appropriate cell fractions to the mitochondrial compartment. Removal of the mitochondrial targeting sequence abolished the competence of HFA1 DNA to complement hfal null mutants. Conversely and in contrast to the intact HFA1 sequence, the signal sequence-free HFA1 gene complemented the mutational loss of cytoplasmic acetyl-CoA carboxylase. Expression of HFA1 under the control of the ACC1 promoter restored cellular ACC activity in ACC1-defective yeast mutants to wild type levels. From this finding, it is concluded that HFA1 encodes a specific mitochondrial acetyl-CoA carboxylase providing malonyl-CoA for intraorganellar fatty acid and, in particular, lipoic acid synthesis.     

Functional specificity of the mitochondrial DnaJ protein, Mdj1p, in Saccharomyces cerevisiae

Inactivation of the gene for the mitochondrial DnaJ homolog, Mdj1p, in Saccharomyces cerevisiae results in temperature sensitivity and the loss of respiratory activity; the latter phenotype has been attributed to the loss of mitochondrial DNA. To investigate the functional specificity of Mdj1p, non-mitochondrial DnaJ proteins were targeted to mitochondria and tested for their ability to substitute for Mdj1p. The tested DnaJ proteins were able to complement the two Mdj1p-linked phenotypes, i.e., respiratory activity and growth at 37 °C, to different extents, ranging from full to very poor complementation. All DnaJ homologs ensured faithful propagation of the mitochondrial genome. N-terminal fragments of Mdj1p and Escherichia coli DnaJ comprising the well-characterized J domain partially substituted for Mdj1p. As the only hitherto known function of the N-terminal fragment is modulation of the substrate binding activity of the cognate Hsp70, we conclude that both Mdj1p-linked phenotypes – maintenance of respiratory activity and the ability to grow at elevated temperature – involve a mitochondrial Hsp70 partner protein. Received: 8 October 1999 / Accepted: 21 January 2000     

POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA

In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD⁺, with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8^m reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases.     

The processing alpha1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum.

The yeast alpha1,2-mannosidase Mns1p is involved in N-linked oligosaccharide processing in Saccharomyces cerevisiae by converting Man9GlcNAc2 to a single isomer of Man8GlcNAc2. alpha1,2-Mannosidase is a 63 kDa type II resident membrane protein of the endoplasmic reticulum that has none of the known endoplasmic reticulum localization signals (HDEL/KDEL, KKXX, or RRXX). Using antibodies against recombinant alpha1,2-mannosidase, indirect immunofluorescence showed that alpha1,2-mannosidase localization is abnormal in rer1 cells and that the alpha1,2-mannosidase localizes in the vacuoles of rer1/deltapep4 cells whereas in wild-type and deltapep4 cells it is found in the endoplasmic reticulum. 35S-labeled cell extracts were subjected to double immunoprecipitation, first with antibodies to alpha1,2-mannosidase, then with either alpha1,2-mannosidase antibodies or antibodies to alpha1,6-mannose residues added in the Golgi. The labeled proteins were examined by autoradiography after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A significant proportion of the labeled alpha1,2-mannosidase was immunoprecipitated by alpha1,6-mannose antibodies in wild-type, deltapep4 and rer1/deltapep4 cells with endogenous levels of alpha1,2-mannosidase, and in wild-type, deltapep4, rer1 and rer1/deltapep4 cells overexpressing alpha1,2-mannosidase. The alpha1,2-mannosidase of rer1/deltapep4 cells had a slower mobility on the gels than alpha1,2-mannosidase precipitated from wild-type or deltapep4 cells, indicating increased glycosylation due to transport through the Golgi to the vacuoles. It is concluded that the endoplasmic reticulum localization of alpha1,2-mannosidase in wild-type cells depends on Rer1p for retrieval from an early Golgi compartment.     

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli.

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.     

Fcf1p and Fcf2p are novel nucleolar Saccharomyces cerevisiae proteins involved in pre-rRNA processing

The uncharacterized Saccharomyces cerevisiae proteins Fcf1 and Fcf2, encoded by the ORFs YDR339c and YLR051c, respectively, were identified in a tandem affinity purification experiment of the known 40S factor Faf1p. Most of the proteins associated with TAP-Faf1p are trans-acting factors involved in pre-rRNA processing and 40S subunit biogenesis, in agreement with the previously observed role of Faf1p in 18S rRNA synthesis. Fcf1p and Fcf2p are both essential and localize to the nucleolus. Depletion of Fcf1p and Fcf2p leads to a decrease in synthesis of the 18S rRNA, resulting in a deficit in 40S ribosomal subunits. Northern analysis indicates inefficient processing of pre-rRNA at the A(0), A(1), and A(2) cleavage sites.